The Protein Facility of the Netherlands Cancer Institute (NKI) in Amsterdam has a strong record in structural biology using macromolecular X-ray crystallography, Electron Microscopy and sample preparation, and is also extremely well-equipped for studying molecular biophysics.
We are very experienced in protein production using insect and mammalian cells, and assist users with the design of their expression construct in terms of solubility, dopmain boundaries, etc. We offer a variety of exprssion vectors, including a set of LIC vectors (Ligand Independent Cloning) for protein expression in insect cells and mammalian cells. For expression in insect cells we have SF9 and SF21 systems for expression of intracellular and extracellular proteins.Our mammalian expression system has been optimized for HEK-293T cells, and we also have the glycosylation free, gnT1-deficient HEK293S variety.
Protein expression in bacteria (E. coli) is beyond the scope of the facility!
Depending on the project, the platform for expression will be chosen. Proteins can be expressed in E. coli, Insect cells and mammalian cells. For each of the systems we can do small scale (few ml) as well as large scale (few liters) production. For insect cell expression SF9 and SF21 cells are used for expression of intracellular and extracellular proteins. The cells are grown in suspension cultures in volumes up to 500 ml/flask. We use Baculovirus technology for over-expression of proteins in insect cells.
Depending on the project, the platform for expression will be chosen. Proteins can be expressed in E. coli, Insect cells and mammalian cells. For each of the systems we can do small scale (few ml) as well as large scale (few liters) production. Our mammalian expression system has currently been optimized for HEK-293T cells and is typically used for expression of extracellular (secreted) proteins. These cells can be cultured in flasks, dishes or rollerbottles. A glycosylation deficient strain - gnT1-deficient HEK293S - is also available for production of eukaryotic proteins that would benefit from removal of glycosyl chains (e.g. for crystallisation purposes). The use of other cells for expression can be discussed.